PTPN22 Is Genetically Associated with Risk of Generalized Vitiligo, but CTLA4 Is Not

ORIGINAL ARTICLE

PTPN22 Is Genetically Associated with Risk of Generalized Vitiligo, but CTLA4 Is Not Greggory S. LaBerge1, Dorothy C. Bennett2, Pamela R. Fain1,3 and Richard A. Spritz1,4 Generalized vitiligo is an acquired, multifactorial, polygenic disease in which depigmented spots of skin, overlying hair, and mucus membranes result from autoimmune-mediated loss of melanocytes from affected areas. We examined single-nucleotide polymorphisms (SNPs) in the PTPN22 and CTLA4 genes in 126 Caucasian families with multiple cases of generalized vitiligo and associated autoimmune diseases, using a family-based association study design. The PTPN22 1858T allele of SNP rs2476601 is significantly associated both with generalized vitiligo and with an expanded autoimmunity phenotype. Individuals carrying the PTPN22 1858T allele had an allelic odds ratio (OR) of 2.16 for generalized vitiligo and a genotypic OR of 2.35 as C/T heterozygotes. Similarly, individuals carrying the PTPN22 1858T allele had an allelic OR of 2.05 for the expanded autoimmunity phenotype, and a genotypic OR of 2.19 for C/T heterozygotes. Examination of five SNPs in the CTLA4 gene (rs1863800, rs231775, rs3087243, rs11571302, rs11571297, rs10932037) in the same 126 families yielded no evidence of allelic or genotypic association with either generalized vitiligo or the expanded autoimmune phenotype. These results implicate PTPN22 in mediating susceptibility to generalized vitiligo and associated autoimmune diseases, but do not support a role for CTLA4. Journal of Investigative Dermatology (2008) 128, 1757–1762; doi:10.1038/sj.jid.5701233; published online 17 January 2008

INTRODUCTION Generalized vitiligo is an acquired, non-contagious disorder in which progressive, patchy loss of pigmentation of skin, overlying hair, and mucous membranes results from loss of melanocytes from the involved areas (reviewed in Hann and Nordlund, 2000; Nordlund et al., 2006). Known for thousands of years because of its visually evident phenotype, vitiligo is the most common pigmentation disorder, affecting about 0.4% of Caucasians (Howitz et al., 1977) and occurring with generally similar frequency in other populations (for example, Das et al., 1985a; Lu et al., 2007). Several different etiologic hypotheses have been suggested for generalized vitiligo (reviewed in Nordlund et al., 2006), the most compelling of which involves a combination of unknown environmental and genetic factors interacting to contribute to autoimmune melanocyte destruction.

1

Human Medical Genetics Program, University of Colorado Denver, Anschutz Medical Campus, Aurora, Colorado, USA; 2Division of Basic Medical Sciences, St George’s, University of London, London, UK; 3Barbara Davis Center for Childhood Diabetes, University of Colorado Denver, Anschutz Medical Campus, Aurora, Colorado, USA and 4Department of Pediatrics, University of Colorado Denver, Anschutz Medical Campus, Aurora, Colorado, USA Correspondence: Professor Richard A. Spritz, Human Medical Genetics Program, University of Colorado Denver, Anschutz Medical Campus, PO Box 6511, MS 8300, Aurora, Colorado 80045 USA. E-mail: [email protected] Abbreviations: CI, confidence interval; LD, linkage disequilibrium; OR, odds ratio; SNP, single-nucleotide polymorphism

Received 15 August 2007; revised 20 November 2007; accepted 26 November 2007; published online 17 January 2008

& 2008 The Society for Investigative Dermatology

Most cases of generalized vitiligo occur sporadically, although about 15–20% of patients report one or more affected first-degree relatives. Typically, familial aggregation of generalized vitiligo cases occurs in a non-Mendelian pattern that is suggestive of polygenic, multifactorial inheritance (Mehta et al., 1973; Carnevale et al., 1980; Hafez et al., 1983; Das et al., 1985a, b; Majumder et al., 1988, 1993; Bhatia et al., 1992; Nath et al., 1994; Alkhateeb et al., 2003; LaBerge et al., 2005; Sun et al., 2006), and formal genetic segregation analyses have indicated that multiple major loci contribute to vitiligo susceptibility in a complex interactive manner (Majumder et al., 1993; Nath et al., 1994; Sun et al., 2006). Furthermore, patients with generalized vitiligo are also at increased risk of developing other autoimmune diseases, particularly autoimmune thyroid disease (Graves’ disease and autoimmune hypothyroidism), rheumatoid arthritis, psoriasis, latent autoimmune diabetes of adults, pernicious anemia, Addison’s disease, and systemic lupus erythematosus (Alkhateeb et al., 2003; LaBerge et al., 2005). These same diseases also occur at increased frequencies in vitiligo patients’ first-degree relatives, suggesting that susceptibility to this group of autoimmune diseases is genetically determined. Genetic linkage and association studies have implicated a number of different genes in susceptibility to generalized vitiligo (reviewed in Spritz, 2007), including several that are thought to play primary roles in the development of autoimmunity. These include the major histocompatibility complex (for example, Foley et al., 1983; Finco et al., 1991; Orecchia et al., 1992; Ando et al., 1993; Schallreuter et al., 1993; al-Fouzan et al., 1995; Zamani et al., 2001; Arcoswww.jidonline.org 1757

GS LaBerge et al. Association Study of PTPN22 and CTLA4 with Vitiligo

Burgos et al., 2002; Tastan et al., 2004; Fain et al., 2006; Xia et al., 2006; Liu et al., 2007), CTLA4 (Kemp et al., 1999; Blomhoff et al., 2005, Itirli et al., 2005), and PTPN22 (Canton et al., 2005), and NALP1 (Jin et al., 2007a, b). Association of vitiligo with PTPN22 and CTLA4 is based on small case–control studies, a study design that is notoriously subject to both false-positive and false-negative errors due to population admixture and stratification (Hirschhorn et al., 2002). To more rigorously test the association of vitiligo with variation in PTPN22 and CTLA4, we carried out family-based association analyses of CTLA4 and PTPN22 single-nucleotide polymorphisms (SNPs) in 126 Caucasian families with multiple cases of generalized vitiligo, which included the same families in which we previously showed association to HLA markers (Fain et al., 2006) and NALP1 (Jin et al., 2007a). RESULTS

Table 1. Results of Hardy–Weinberg equilibrium (HWE) analysis of SNP genotypes among founders from 126 multiplex vitiligo-autoimmune disease families SNP

1758 Journal of Investigative Dermatology (2008), Volume 128

HWE P-value

PTPN22 rs2476601

T/T 2 C/T 34 C/C 104

0.9170

rs1863800

T/T 19 C/T 74 C/C 45

0.4210

rs231775

G/G 41 A/G 73 A/A 25

0.7492

rs3087243

A/A 23 A/G 76 G/G 40

0.4304

rs11571302

A/A 24 A/C 75 C/C 38

0.4602

rs11571297

G/G 29 A/G 75 A/A 34

0.5830

rs10932037

T/T 4 C/T 27 C/C 109

0.3750

CTLA4

Family-based association analysis of PTPN22

We genotyped rs2476601 (PTPN22 risk allele 1858T) in 712 individuals from 126 Caucasian extended families with multiple cases of vitiligo as well as other autoimmune diseases, derived from the United States and the United Kingdom (Alkhateeb et al., 2003; LaBerge et al., 2005; Jin et al., 2007a). SNP rs2476601 was found to be in Hardy– Weinberg equilibrium in founders (individuals with no parents specified in the pedigree) in the 126 study families (Table 1). As shown in Table 2, we found that the high-risk 1858T allele of rs2476601 was significantly associated (P ¼ 0.0048) with generalized vitiligo in the 126 study families. Genotypic analysis showed that the rs2476601 heterozygous C/T genotype was highly associated with vitiligo (P ¼ 0.0012). The PTPN22 high-risk 1858T allele showed allelic association with generalized vitiligo [P ¼ 0.024, odds ratio (OR) 2.16 (95% confidence interval (CI) 1.22–3.82)] (Table 2) from conditional logistic regression analysis. We also tested an expanded autoimmunity phenotype that included all of the autoimmune diseases with which vitiligo is epidemiologically associated (autoimmune thyroid disease, rheumatoid arthritis, psoriasis, adult-onset autoimmune diabetes mellitus, pernicious anemia, systemic lupus erythematosus, Addison’s disease), considering any individual with either vitiligo or any other of these autoimmune diseases as ‘‘affected’’. As shown in Table 2, the rs2476601 1858T allele also showed allelic association (P ¼ 0.0042) with this expanded autoimmunity phenotype, which was confirmed by conditional logistic regression analysis [P ¼ 0.03, OR 2.05 (95% CI 1.19–3.53)]. Genotypic analysis, presented in Table 3, showed that the rs2476601 C/T genotype is significantly associated with the expanded autoimmunity phenotype (P ¼ 0.003), although association with the T/T genotype was not significant (P ¼ 0.750). Conditional logistic regression analysis provided very similar results, showing significant association of the C/T heterozygote with the autoimmune disease phenotype [P ¼ 0.03, OR 2.19 (95% CI 1.20–3.97)], but non-significant association of the T/T homozygote [P ¼ 0.14, OR 3.22 (95% CI 0.68–15.27)].

Observed genotypes in founders

SNP, single-nucleotide polymorphism.

Family-based association analysis of CTLA4

We also examined six SNPs in the CTLA4 gene (rs1863800, rs231775, rs3087243, rs11571302, rs11571297, rs10932037) for association with generalized vitiligo and the expanded vitiligo-associated autoimmune phenotype in these families. None of the CTLA4 SNPs tested exhibited either allelic (Table 2) or genotypic (Table 3) association with either generalized vitiligo or with the expanded autoimmunity phenotype. All but one of these SNPs clustered into a single linkage disequilibrium (LD) block (data not shown), and no multiple testing corrections were applied to the nominal P-value threshold (P ¼ 0.05) due to lack of any apparent association with disease. DISCUSSION We have carried out family-based association analyses of PTPN22 and CTLA4 SNPs in 126 Caucasian extended families with multiplex cases of vitiligo as well as other autoimmune diseases, confirming the association of generalized vitiligo with the functional PTPN22 1858T (R620W)variant allele (OR 2.16, 95% CI 1.22–3.82) and the heterozygous C/T genotype (OR 2.35, 95% CI 1.25–4.43). Furthermore, we find that the PTPN22 1858T allele and C/T genotype are also associated with an expanded autoimmunity phenotype (allelic OR 2.05, 95% CI 1.19–3.53; genotypic OR 2.19, 95% CI 1.20–3.97) that includes generalized vitiligo or

GS LaBerge et al. Association Study of PTPN22 and CTLA4 with Vitiligo

Table 2. Allelic association analyses of PTPN22 and CTLA4 SNPs FBAT1(Pvalue)

Regression on alleles (P-value)

Odds ratio (95% CI)

0.00482

0.0242

2.16 (1.22–3.82)

rs1863800

0.3742

0.495

0.89 (0.63–1.24)

rs231775

0.5440

0.592

1.09 (0.79–1.52)

rs3087243

0.3518

0.683

0.934 (0.67–1.30)

rs11571302

0.3628

0.930

0.986 (0.71–1.36)

rs11571297

0.3942

0.858

1.03 (0.74–1.44)

rs10932037

0.8688

1.00

1.00 (0.61–1.65)

0.00422

0.032

2.05 (1.19–3.53)

rs1863800

0.2907

0.305

0.85 (0.62–1.16)

rs231775

0.4198

0.310

0.85 (0.62–1.16)

rs3087243

0.2460

0.370

0.87 (0.63–1.18)

rs11571302

0.2655

0.578

0.92 (0.67–1.25)

rs11571297

0.2920

0.465

1.13 (0.82–1.56)

rs10932037

0.8858

0.809

0.94 (0.59–1.52)

SNP Vitiligo PTPN22 rs2476601 CTLA4

Expanded autoimmunity phenotype PTPN22 rs2476601 CTLA4

CI, confidence interval; FBAT, family-based association test; SNP, singlenucleotide polymorphism. 1 P-values based on FBAT additive model. 2 P-values corrected for three independent tests: two LD blocks in CTLA4 and one SNP in PTPN22.

any of the other autoimmune diseases with which vitiligo is epidemiologically associated (Alkhateeb et al., 2003; LaBerge et al., 2005). These findings strongly support a causal role for the PTPN22 1858T allele of rs2476601 (or another variant with which 1858T is in close LD) in susceptibility to both generalized vitiligo and these other autoimmune diseases. We found no association of the PTPN22 rs2476601 homozygous T/T genotype with either generalized vitiligo or the expanded autoimmune phenotype, most likely due to limited power resulting from the low frequency of the T allele (0.13 in this study) and the consequent small number of T/T homozygotes observed (8 of 712 individuals genotyped). However, our analyses of the CTLA4 SNPs showed no apparent allelic or genotypic association with either generalized vitiligo or the expanded autoimmunity phenotype. These results are in contrast with previous reports of genetic association of CTLA4 markers with vitiligo based on small case–control studies (Kemp et al., 1999; Blomhoff et al., 2005; Itirli et al., 2005). Kemp et al. (1999) reported the association of the 106-bp allele of a microsatellite polymorphism in the CTLA4 3’-untranslated region with vitiligo (especially autoimmune-associated) in 74 European Caucasian patients versus 173 controls. The same group (Blomhoff et al., 2005) subsequently reported the association

of CTLA4 SNPs MH30 (23 kb 50 of the CTLA4 gene), rs3087243, rs11571302, and rs7565213 with vitiligo in 27 European Caucasian patients with associated autoimmune disease versus 140 controls; it is not clear whether the case and control cohorts in these two studies were independent. Itirli et al. (2005) reported the association of the 96-bp allele of the same CTLA4 microsatellite in 36 Turkish patients versus 100 controls. It may be that these previous ‘‘associations’’ represent spurious false-positive results, due to either occult population stratification (reviewed in Hirschhorn et al., 2002) or the very small sample size and highly unbalanced numbers of cases and controls in both studies. Furthermore, Blomhoff et al. (2005) did not correct for multiple testing, which would have rendered the reported CTLA4 associations insignificant. Itirli et al. (2005) reported significance based on the observation of only four vitiligo patients versus two controls with the 96-bp allele, whereas the 112-bp allele, which showed the largest difference in allele frequency between cases and controls, was not significant, indicating that ‘‘significance’’ of the 96-bp allele almost certainly represents statistical fluctuation due to the very small number of observations. PTPN22 and CTLA4 are thought to function as general autoimmunity susceptibility loci (Brand et al., 2005; Gregersen et al., 2006). The PTPN22 1858T variant results in an arginine to tryptophan substitution that disrupts interaction between Lyp and Csk protein tyrosine kinases, dis-inhibiting T-cell activation (Siminovitch, 2004) and perhaps thereby increasing susceptibility to autoimmune disease (Brand et al., 2005). The PTPN22 1858T variant has been associated with type 1 diabetes, rheumatoid arthritis, systemic lupus erythematosus, Graves’ disease (Bottini et al., 2004; Siminovitch, 2004; Criswell et al., 2005; Zhernakova et al., 2005), and vitiligo (Canton et al., 2005). In contrast, studies of patients with psoriasis and multiple sclerosis showed no association with the PTPN22 risk allele in two family-based studies (Criswell et al., 2005; Nistor et al., 2005), suggesting that PTPN22 may not truly be associated with these diseases. Our study utilized a family-based design to test association of PTPN22 and CTLA4 with generalized vitiligo, an approach that is more rigorous than previous case–control studies of these genes in vitiligo. Our findings demonstrate that PTPN22 can be included along with HLA and NALP1 among those genes confirmed to play a role in polygenic susceptibility to generalized vitiligo, but do not support a role for CTLA4. MATERIALS AND METHODS Subjects Peripheral blood or saliva samples were obtained from 712 individuals from 126 extended families with multiplex cases of generalized vitiligo as well as other autoimmune diseases, derived from the United States and United Kingdom. All families were of Caucasian origin (as self-reported) and had two or more family members with generalized vitiligo and at least one family member with one or more other autoimmune disease with which generalized vitiligo is epidemiologically associated (autoimmune thyroid disease, latent autoimmune diabetes in adults, psoriasis, pernicious anemia, systemic lupus erythematosus, rheumatoid arthritis, www.jidonline.org 1759

GS LaBerge et al. Association Study of PTPN22 and CTLA4 with Vitiligo

Table 3. Genotypic association analyses of PTPN22 and CTLA4 SNPs

SNP

FBAT1 Genotype (P-value)

Regression on genotypes (P-value)

Odds ratio (95% CI)

Vitiligo PTPN22 rs2476601

T/T

0.814

0.126

3.42 (0.71–16.54)

C/T

0.00122

0.0242

2.35 (1.25–4.43)

C/C

0.999

Referent

CTLA4 rs1863800

rs231775

rs3087243

rs11571302

rs11571297

rs10932037

T/T

0.807

0.542

0.797 (0.385–1.65)

C/T

0.488

0.576

0.886 (0.579–1.35)

C/C

0.327

Referent

A/A

0.408

0.592

0.830 (0.419–1.64)

A/G

0.563

0.762

1.08 (0.657–1.77)

G/G

0.944

Referent

A/A

0.732

0.652

0.854 (0.430–1.70)

A/G

0.552

0.922

0.978 (0.623–1.53)

G/G

0.309

Referent

A/A

0.448

0.752

0.892 (0.440–1.81)

A/C

0.989

0.523

1.16 (0.738–1.82)

C/C

0.528

Referent

G/G

0.567

0.706

1.15 (0.553–2.40)

A/G

0.957

0.325

1.36 (0.740–2.48)

A/A

0.473

Referent

T/T

0.848

1.00

1.00 (0.234–4.28)

C/T

0.751

1.00

1.00 (0.572–1.75)

C/C

0.799

Referent

Expanded autoimmunity phenotype PTPN22 rs2476601

T/T

0.750

0.140

3.22 (0.68 –15.27)

C/T

0.0032

0.032

2.19 (1.20 – 3.97)

C/C

0.999

Referent

CTLA4 rs1863800

rs231775

rs3087243

rs11571302

rs11571297

rs10932037

T/T

0.636

0.379

0.734 (0.368–1.46)

C/T

0.553

0.358

0.829 (0.555–1.24)

C/C

0.299

Referent

A/A

0.187

0.310

0.712 (0.369–1.37)

A/G

0.271

0.933

1.02 (0.638–1.63)

G/G

0.896

Referent

A/A

0.557

0.372

0.748 (0.394–1.42)

A/G

0.619

0.550

0.876 (0.568–1.35)

G/G

0.256

Referent

A/A

0.371

0.506

0.801 (0.416–1.54)

A/C

0.992

0.955

1.01 (0.656–1.56)

C/C

0.426

Referent

G/G

0.454

0.418

1.32 (0.672–2.60)

A/G

0.987

0.334

1.31 (0.759–2.24)

A/A

0.403

Referent

T/T

0.784

0.942

0.948 (0.223–4.02)

C/T

0.965

0.789

0.930 (0.549–1.58)

C/C

0.955

Referent

CI, confidence interval; FBAT, family-based association test; SNP, singlenucleotide polymorphism. 1 P-values based on FBAT genotype model. 2 P-values corrected for 3 independent tests: 2 LD blocks in CTLA4 and 1 SNP in PTPN22.

1760 Journal of Investigative Dermatology (2008), Volume 128

Addison’s disease; Alkhateeb et al., 2003; LaBerge et al., 2005). Diagnostic criteria for generalized vitiligo were consistent with those of the Vitiligo European Task Force (Taı¨eb and Picardo, 2007). Exclusion criteria were atypical lesion distribution, congenital or static skin depigmentation (for example, birthmarks, piebaldism, Waardenburg syndrome), depigmentation secondary to the use of melanocytotoxic chemicals, inflammatory skin diseases (systemic lupus erythematosus, lichen planus, psoriasis), and post-infectious or post-traumatic causes. All available affected and unaffected family members completed a clinical history questionnaire reporting vitiligo age of onset and course of treatment, and a checklist of approximately 50 autoimmune and autoinflammatory diseases. Each vitiligo patient completed a skin-lesion map. All data were reviewed by study investigators and staff, and individuals in whom diagnoses were uncertain based on standard diagnostic criteria (Nordlund et al., 2006) were excluded from the study. This study conformed to the Declaration of Helsinki Principles and was approved by the Colorado Multiple Institutional Review Board and the South East Research Ethics Committee. Written, informed consent was provided by all study participants.

SNP genotyping DNA was isolated from peripheral blood using a genomic DNA purification kit (Purgene, Gentra Systems) or from saliva obtained using the DNA self-collection kit (Oragene; DNA Genotek, Ottawa, Ontario, Canada). Genotyping was carried out for PTPN22rs2476601 (1858C/T), and CTLA4-rs1863800, rs231775 ( þ 49G), rs3087243 (CT60), rs11571302 (JO31), rs11571297 (JO27_1), rs10932037 (CTIC154_1) in 10-ml PCR reaction volumes with 20 ng genomic DNA using standard methods. SNP alleles were detected with the Applied Biosystems (ABI, Foster City, CA) PRISM SNaPshot Multiplex Kit using an ABI 3130 Genetic Analyzer. Automated genotyping was performed using ABI Genemapper version 3.7 software; all genotypes were manually checked for accuracy and allele calling consistency. Mendelian inheritance of all markers was checked through all levels of PedCheck (O’Connell and Weeks, 1998). Haplotype analysis of CTLA4 SNPs was carried out using the error-screening routine of Merlin, version 1.0.0 (Abecasis et al., 2002).

Statistical analyses Deviations from expected Hardy–Weinberg proportions were tested for each SNP in founders (no parents in the pedigree) and in persons not in the lineage in all 126 families using w2-analysis, considering Po0.05 significant. Calculation of LD between CTLA4 SNPs was performed using Haploview software (Barrett et al., 2005), version 3.32, with haplotype blocks being determined by the method of Gabriel et al. (2002). Single-locus association analyses of each SNP were carried out using the family-based association test (Horvath et al., 2001). Conditional logistic regression analysis was carried out using STATA, version 9.2, which uses family-based data to create a matched pseudo case–control data set, test for allelic or genotypic association and estimate an OR for disease risk (Cordell and Clayton, 2002). Nominally significant P-values (o0.05) were corrected for three independent tests based on LD analysis, using Haploview (two LD blocks defined in the CTLA4 gene and one SNP in PTPN22).

GS LaBerge et al. Association Study of PTPN22 and CTLA4 with Vitiligo

CONFLICT OF INTEREST The authors state no conflict of interest.

ACKNOWLEDGMENTS We thank the many families who participated in this study, and the UK Vitiligo Society, the US National Vitiligo Foundation, and Vitiligo Support International for their enthusiastic help in family ascertainment. We thank Anita Amadi-Myers and Paulene Holland for their invaluable assistance. This work was supported by grants AR45584, AI46374, and DK57538 from the National Institutes of Health.

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